Treating Tumors Using TTFields Combined with a PARP Inhibitor

ABSTRACT

Tumor-Treating Fields elicit a conditional vulnerability to PARP inhibitors (e.g., Olaparib) in certain cancer cells such as non-small cell lung cancer (NSCLC) cell lines. This conditional vulnerability is exploited in a method of killing cancer cells that comprises delivering a PARP inhibitor to the cancer cells and applying an alternating 80-300 kHz electric field to the cancer cells. At least a portion of the applying step is performed simultaneously with at least a portion of the delivering step. In some embodiments, an additional step of treating the cancer cells with a radiation therapy is added to the method. In some embodiments, the frequency of the alternating electric field is between 100 and 200 kHz.

BACKGROUND

Lung cancer is the second most prevalent cancer and the leading cause ofcancer-related death in the United States. Non-small cell lung cancer(NSCLC) is the most prevalent type, accounting for ˜80% of new cases. Aplethora of treatment options exist including surgical resection,chemotherapy, radiation therapy, and immunotherapy. Five-year survivalrates for patients with stage I and II NSCLC are ˜50% and 30%,respectively. However, despite this myriad of options, 5-year survivalrates for patients with late stage IIIA, IIIB and IV are 14%, 5% and 1%,respectively, highlighting the need for novel treatment modalities thatcan be utilized alone or in combination with conventional therapies toincrease survival rates.

The advent of Tumor-Treating Fields (TTFields), a novel physicaltreatment modality, has been effective for the treatment of solid,therapy-resistant primary and recurrent tumors. TTFields electrodes arenon-invasive and deliver a low-intensity (e.g., 1-3 V/cm) intermediatefrequency (e.g., 100-300 kHz) alternating electric field across thetumor bed. TTFields create a heterogeneous intracellular environmentthat induces a dielectrophoretic movement of polar molecules toward theregion of higher field intensity, effectively preventing polymerizationand other critical biochemical functions. As such, TTFieldspreferentially target cancer cells through the exploitation of cellproliferation, effectively sparing non-dividing normal cells. Inaddition, TTFields do not stimulate nerves and muscle because of theirhigh frequency, and do not generate high levels of heating because oftheir low intensity. The FDA has approved Optune (NovoCure), a TTFieldsgenerating transducer array, for the treatment of recurrent and newlydiagnosed glioblastoma (GBM) in combination with temozolomide.

SUMMARY OF THE INVENTION

One aspect of the invention is directed to a first method of killingcancer cells. The first method comprises delivering a PARP inhibitor tothe cancer cells; and applying an alternating electric field to thecancer cells. The alternating electric field has a frequency between 80and 300 kHz, and at least a portion of the applying step is performedsimultaneously with at least a portion of the delivering step.

In some embodiments of the first method, the PARP inhibitor comprisesOlaparib. In some embodiments of the first method, the cancer cells areNSCLC cells. In some embodiments of the first method, the applying stephas a duration of at least 72 hours. In some embodiments of the firstmethod, the frequency of the alternating electric field is between 100and 200 kHz.

In some embodiments of the first method, the PARP inhibitor is deliveredto the cancer cells at a therapeutically effective concentration, andthe alternating electric field has a field strength of at least 1 V/cmin at least some of the cancer cells. In some of these embodiments, theapplying step has a duration of at least 72 hours and the frequency ofthe alternating electric field is between 100 and 200 kHz. In some ofthese embodiments, the PARP inhibitor comprises Olaparib. In some ofthese embodiments, the cancer cells are NSCLC cells.

Another aspect of the invention is directed to a second method ofkilling cancer cells. The second method comprises delivering a PARPinhibitor to the cancer cells; applying an alternating electric field tothe cancer cells; and treating the cancer cells with a radiationtherapy. The alternating electric field has a frequency between 80 and300 kHz, and at least a portion of the applying step is performedsimultaneously with at least a portion of the delivering step.

In some embodiments of the second method, the PARP inhibitor comprisesOlaparib. In some embodiments of the second method, the cancer cells areNSCLC cells. In some embodiments of the second method, the PARPinhibitor is delivered to the cancer cells at a therapeuticallyeffective concentration, and the alternating electric field has a fieldstrength of at least 1 V/cm in at least some of the cancer cells.

In some embodiments of the second method, the PARP inhibitor isdelivered to the cancer cells at a therapeutically effectiveconcentration, and the alternating electric field has a field strengthof at least 1 V/cm in at least some of the cancer cells, and thedelivering, applying, and treating steps are repeated at least fivetimes. In some of these embodiments, each repetition of the treatingstep comprises delivering at least 2 Gy of radiation to a target area.In some of these embodiments, each repetition of the treating stepcomprises delivering at least 4 Gy of radiation to a target area. Insome of these embodiments, the frequency of the alternating electricfield is between 100 and 200 kHz. In some of these embodiments, thetreating step is performed immediately after the applying step in eachrepetition. In some of these embodiments, the applying step is performedimmediately after the treating step in each repetition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the fraction of cells surviving TTFields treatment fordifferent NSCLC cell lines.

FIGS. 2A-D show the impact of TTFields treatment on NSCLC cells in theG2/M phase, the S-phase, the sub-G1 phase, and the G1 phase,respectively.

FIG. 3A shows z-scores and P-values of BRCA1 pathway gene expressionalong with the relevant pathway gene names.

FIG. 3B depicts immunoblots of representative BRCA1 pathway genesresulting from TTFields treatment.

FIG. 4 depicts a quantification of immunoblots indicating that certainprotein levels were downregulated after exposure to TTFields.

FIG. 5A shows how exposure to TTFields changes the mean number oflocalized 53BP1 and γ-H2AX foci for different cell lines.

FIG. 5B shows the change in the mean number of γ-H2AX foci over timewith TTFields and 2 Gy of radiation for different cell lines.

FIG. 5C shows the mean value for residual γ-H2AX foci and localized53BP1 and γ-H2AX foci for different cell lines after TTFields exposurein combination with radiation.

FIG. 5D shows the induction of chromatid-type aberrations in differentNSCLC lines after TTFields exposure in combination with radiation.

FIG. 5E shows the frequency of chromosome-type aberrations afterTTFields exposure in combination with radiation.

FIG. 6 depicts an evaluation of the radiosensitization effect ofTTFields in combination with IR.

FIG. 7 depicts baseline growth curves for exposing various NSCLC celllines to different concentrations of Olaparib without TTFields.

FIG. 8 depicts clonogenic assays showing the combination effect fordifferent combinations of various concentrations of Olaparib andTTFields.

FIGS. 9A and 9B depict clonogenic assays showing the combination effectfor different combinations of TTFields, RT dosage, and Olaparib forH1299 and H157 cell lines, respectively.

FIG. 10 depicts data showing that TTFields inhibit the increase inlength of newly replicated DNA fibers.

FIG. 11 shows that TTFields cause replication fork stress which resultsin R loop formation.

Various embodiments are described in detail below with reference to theaccompanying drawings.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Tumor-Treating Fields Elicit a Conditional Vulnerability to IonizingRadiation (IR) and PARP Inhibitors Via the Downregulation of BRCA1Signaling and Reduced DNA Double-Strand Break Repair Capacity inNon-Small Cell Lung Cancer Cell Lines: The use of tumor-treating fields(TTFields) has revolutionized the treatment of recurrent and newlydiagnosed glioblastoma (GBM). TTFields are low-intensity, intermediatefrequency, alternating electric fields that are applied to tumor regionsand cells using non-invasive arrays. The predominant mechanism by whichTTFields are thought to kill tumor cells is the disruption of mitosis.Using five non-small cell lung cancer (NSCLC) cell lines, the inventorsfound that there is a variable response in cell proliferation and cellkilling between these NSCLC cell lines that was independent of p53status. TTFields treatment increased the G2/M population, with aconcomitant reduction in S-phase cells followed by the appearance of asub-G1 population indicative of apoptosis. Temporal changes in geneexpression during TTFields exposure was evaluated to identify molecularsignaling changes underlying the differential TTFields response. Themost differentially expressed genes were associated with the cell cycleand cell proliferation pathways. However, the expression of genes foundwithin the BRCA1 DNA-damage response were significantly downregulated(P<0.05) during TTFields treatment. DNA double-strand break (DSB) repairfoci increased when cells were exposed to TTFields as did the appearanceof chromatid-type aberrations, suggesting an interphase mechanismresponsible for cell death involving DNA repair. Exposing cells toTTFields immediately following ionizing radiation resulted in increasedchromatid aberrations and a reduced capacity to repair DNA DSBs, whichmay be responsible for at least a portion of the enhanced cell killingseen with the combination. These findings suggest that TTFields induce astate of ‘BRCAness’ leading to a conditional susceptibility resulting inenhanced sensitivity to ionizing radiation and supports the use ofTTFields as a combined modality therapy with radiation, PARP inhibitors,or other DNA-damaging agents.

TTFields are known to decrease cellular proliferation and induceabortive apoptosis in dividing cancer cells across a variety of humanand rodent tumor cell lines. Prevention of proper formation of themitotic spindle apparatus and the activation of the mitotic spindlecheckpoint has been proposed as the mechanism by which TTFields killdividing cells. Specifically, TTFields exposure leads to microtubuledepolymerization and the mislocalization of septin. This results inplasma membrane instability and blebbing that disrupts cytokinesis,leading to abnormal chromosome segregation, aberrant mitotic exit andproduction of deranged cells that subsequently undergo apoptosis.

In the context of cancer therapy, TTFields has been shown to enhance theefficacy of numerous chemotherapeutic agents when used in combinationsuch as paclitaxel and doxorubicin in multidrug-resistant cancercells—without increasing the intracellular accumulation of the drugs;decreased cellular proliferation, survival and the percentage of G2/Mpopulations; enhanced the efficacy of chemotherapy in a hamsterpancreatic cancer model; decreased cellular proliferation and enhancedthe efficacy of pemetrexed, cisplatin and paclitaxel in NSCLC cells bothin vitro and in vivo. In addition to its efficacy in treating primarytumors, TTFields have also demonstrated the ability to prevent or delaymetastasis in animal models, a process that may result from enhancementof the antitumor immune response. Furthermore, TTFields apparentlyenhance the efficacy of radiation treatment through the induction ofincreased mitotic abnormalities and induction of DNA damage in GBM.Although these findings collectively support the efficacy of TTFields asan anticancer agent, further mechanistic insights are needed to optimizethe use of TTFields in combination with additional modalities includingradiation therapy and PARP inhibitors.

Using a panel of NSCLC cell lines with different molecular phenotypes,the inventors found that TTFields alone exhibit antiproliferativeeffects and cytotoxicity. The inventors divided NSCLC cells into moreresponsive (H157 and H4006) and less responsive (A549, H129, H1650) celllines based on their degree of responsiveness to TTFields. Consistentwith previous reports, the inventors also observed a time-dependentincrease in the G2/M population upon TTFields exposure; however, thenumber of cells accumulating in G2/M was likely not enough to accountfor the decreased survival seen.

The inventors postulated that TTFields may induce additional mechanismsleading to cell death. To explore this phenomenon and identify novelmechanisms that could be exploited clinically, the inventors performedtemporal gene expression analysis after treating H157, H4006, A549,H1650 and H1299 cell lines with TTFields for up to 48 h. In addition toconfirming previously described mechanisms with the perturbation ofgenes involved in cell cycle regulation and mitosis, the inventors alsoidentified a significant association of differentially expressed genesto the BRCA1 pathway (P<0.05) upon TTFields treatment. This findingsuggests that a novel mechanism involving DNA repair and/or DNAreplication may contribute to TTFields induced cell killing other thanthe reported abortive mitosis cell death mechanism. TTFields aloneelevated the frequency of chromatid-type aberrations and induced γ-H2AXfoci, in addition to slowing the repair of ionizing radiation(IR)-induced double-strand breaks (DSBs).

The inventors recognized that TTFields sensitized NSCLC cells to IR anddecreased the surviving fraction at 2 and 4 Gy. The effect was at leastadditive and, in some cases, synergistic. Taken together, these resultshighlight a previously unknown mechanism for TTFields-induced cellkilling, and also suggest that TTFields may establish a ‘conditionalvulnerability’ resulting from an induced state of ‘BRCAness’ effectivelysensitizing cells to IR and opening new avenues for combination therapywith DNA-damaging agents and other agents such as PARP inhibitors.

Results

TTFields Reduce NSCLC Cell Proliferation: Previous studies havedemonstrated that TTFields exhibit optimal effectiveness in a cellline-specific manner. To determine the optimal frequency to maximizegrowth inhibition, cells were treated at different frequencies rangingfrom 100 to 300 kHz. Cell counts were taken every 24 h for up to 72 hwithin a panel of NSCLC cell lines. Table 1 lists the NSCLC cell linesutilized in this study, and for each of those cell lines: theirstandardized optimized frequency at which maximal growth inhibition wasobserved, average percentage of growth inhibition at the optimizedfrequency after 72 h of TTFields exposure, genetic backgroundinformation (i.e., their p53 and KRAS mutation status), and the cellcycle-doubling time (N=3). Further experimentation was carried out atthe optimal frequencies listed in Table 1. As reflected in Table 1,while TTFields reduced the growth in all the cell lines examined, itsrelative efficacy was lower in the H1650, H1299, and A549 cell lines andhigher in the H157 and H4006 cell lines.

TABLE 1 Standardized TTFields % of growth Doubling Cell frequencyinhibition at P53 KRAS time line (kHz) 72 h status status (h) H157 10072 Mutant Wild type 36 H4006 150 69 SNP MS Wild type 34 A549 200 48 Wildtype Mutant 22 H1299 100 32 Mutant Wild type 20 H1650 100 21 Wild typeWild type 26

TTFields Exposure Causes Cell Death in NSCLC Cell Lines: As growth delayis not the same as cell killing, the inventors examined the ability ofTTFields to induce reproductive cell death using clonogenic survivalassays. TTFields treatment resulted in a significant decrease in cellsurvival in all the cell lines examined, a trend that generallyincreased with the amount of time cells were exposed to TTFields. Morespecifically, FIG. 1 shows the fraction of cells surviving TTFieldstreatment at 24, 48 and 72 h post induction in a panel of NSCLC celllines including H157, H1299, A549, H1650, and H4006. Values arerepresented as the number of colony-forming cells relative to control.Error bars represent the S.E.M. of three separate experiments andasterisks represent values where survival was significantly (P<0.05)decreased. As with the cell growth patterns, H1650, H1299, and A549 wereless responsive and H157 and H4006 more responsive, to TTFields. Thecharacterization of more responsive versus less responsive wasmaintained for all assays in this study.

TTFields Exposure Alters the Cell Cycle Distribution by Enriching theG2/M Population and Generating a Sub-G1 Population: Previous reportshave established that TTFields alter the cell cycle distribution,resulting in an increase in the G2/M phase of the cell cycle withincreasing treatment time in GBM and ovarian cancer cell lines. Theinventors set out to determine whether TTFields induce a similarenrichment of the G2/M population in NSCLC cell lines. To do this, theinventors performed propidium iodide (PI) staining and examined thedistribution of cells throughout the cell cycle using flow cytometry inthe two most responsive cell lines (H157 and H4006) and two of the lessresponsive cell lines (A549 and H1299).

The inventors' experiments revealed that TTFields treatment enriched theG2/M and G0/G1 populations while decreasing the number of S-phase cellsin all cell lines tested. More specifically, FIGS. 2A-D shows thatTTFields treatment resulted in a significant enrichment of NSCLC cellsin the G2/M phase of the cell cycle (FIG. 2A), a decrease in thepercentage of cells in S-phase (FIG. 2B), and resulted in thesignificant induction of a sub-G1 population (FIG. 2C) indicative of anapoptotic cell population. The percentage of cells in the G1 phase isprovided in FIG. 2D for completeness. Error bars represent the S.E.M. ofthree separate experiments and asterisks represent significant changes(P<0.05) in cell count percentage at a given time point and for a givencell line.

That TTFields generated a sub-G1 population gives strong, albeit notdefinitive, evidence for an apoptotic cell population. These changes incell cycle distribution are likely not sufficient to account for theamount of cell death observed with TTFields application. The inventorspostulated that additional mechanism(s), aside from cell cycleperturbation and abortive apoptosis following mitosis, must becontributing to TTFields-induced cell death.

TTFields Induce Global Gene Expression Changes: To explore alternativepotential mechanisms for TTFields-induced cell death, the inventorsperformed gene expression analysis on a panel of NSCLC cells exposed toTTFields for up to 48 h. Differential gene expression after TTFieldsexposure was examined using significance analysis of microarray (SAM)time course analysis. By normalizing TTFields-induced gene expression tothe baseline gene expression values for each cell line, the inventorsidentified a 1083 gene (false discovery rate (FDR)<0.01) signature thatsegregates cell lines by response to TTFields exposure. Gene expressionanalysis was subsequently performed in more responsive and lessresponsive cell groups, respectively. The analysis suggested that, as aresult of TTFields exposure, the expression of 1039 genes was altered inthe less responsive cell lines and that 628 genes were differentiallyexpressed in the more responsive NSCLC cell lines. More specifically,data depicting supervised clustering using a 1083 gene (FDR<0.01)signature that segregates cell lines by response to TTFields exposurewas obtained. Clustering analysis of differentially expressed genesafter TTFields treatment revealed that 628 genes (FDR<0.05) responded toTTFields in the more responsive cell lines, and 1039 genes (FDR<0.05)responded to TTFields in the less responsive cell lines. Clusteranalysis showed distinct expression profiles that separated the 48 htime point from earlier time points relative to untreated controls inthe more responsive lines as well as in less responsive cell lines.

Ingenuity pathway analysis (IPA) was performed to determine specificcanonical pathways involved in the TTFields responding genes. Thisanalysis identified differentially regulated canonical pathways forTTFields exposure in the more responsive and the less responsive celllines. Downregulation of the BRCA1 DNA damage repair pathway was morepronounced in the more responsive cell lines compared to the lessresponsive cell lines, which was associated with negative z-scores. Theresults suggested that alterations occurred in cell cycle and mitoticregulatory pathways, which is consistent with previous studies, but alsorevealed a significantly downregulated BRCA1 DNA-damage response pathway(P<0.05) with TTFields exposure.

BRCA1 Pathway Genes are Downregulated as a Result of TTFields Treatment:Based on IPA analysis of differentially expressed genes, the inventorstheorized that the activity of the BRCA1 pathway was inhibited in NSCLCcells as a result of TTFields exposure. Although inhibition occurred inboth groups, the inventors observed a stronger inhibition in the moreresponsive cell lines compared to the less responsive cell lines asindicated by the negative z-scores. This is depicted in FIG. 3A, whichshows z-scores and P-values of BRCA1 pathway gene expression along withthe relevant pathway gene names.

Temporal gene expression graphs revealed downregulation of many of theBRCA1 pathway genes in all cell lines. The confirmation of BRCA1 pathwaygene downregulation at the protein level was conducted withimmunoblotting for BRCA1, FANCD2 and FANCA, and FIG. 3B depictsimmunoblots of representative BRCA1 pathway genes demonstrating thedownregulation of BRCA1, FANCD2, and FANCA protein levels resulting fromTTFields treatment at 72 h. FIG. 4 depicts the quantification ofimmunoblots (N=3), which reveals that BRCA1, FANCD2, and FANCA proteinlevels were significantly downregulated in all cell lines at 72 hpost-TTFields exposure, confirming gene expression results.

TTFields Cause DNA Damage, Reduce IR-Induced DNA Repair, and Increasethe Frequency of Chromatid-Type Aberrations: Because TTFields decreasedBRCA1-associated gene expression, the inventors wanted to confirmwhether this resulted in DNA damage induction as a result of TTFieldsexposure alone or whether there would be a reduction in DNA DSB repairkinetics after IR. Exposure to TTFields alone resulted in the formationof γ-H2AX foci, with the mean number of foci per cell increasing as afunction of time, indicating to the inventors that TTFields treatmentalone is capable of causing DNA damage. These findings are depicted inFIG. 5B, which shows the change in the mean number of γ-H2AX foci overtime with TTFields alone (triangle icons) and after receiving 2 Gy(round and square icons), both followed for four different cell linesover 48 h; and in FIG. 5C, which shows the mean value for residualγ-H2AX foci and localized 53BP1 and γ-H2AX foci at 48 h post-IR for allfour cell lines after TTFields exposure in combination with radiation.

An IR exposure of 2 Gy immediately followed by TTFields applicationdecreased the resolution of γ-H2AX foci and colocalized γ-H2AX/53BP1foci, indicating to the inventors that in addition to causing DNA damageTTFields also reduced the repair of IR-induced damage. These findingsare depicted in FIG. 5A, which shows the changes in the mean number oflocalized 53BP1 and γ-H2AX foci for four different cell lines over 48 hof exposure to TTFields; as well as in FIG. 5B. When residual lesions at24 and 48 h were compared, the more responsive cell lines had greaternumbers of residual lesions compared to those cell lines considered asless responsive.

To further confirm the effects of TTFields on DNA damage, the inventorsperformed cytogenetic analysis to validate the findings associated withDNA repair foci at 48 h post irradiation. TTFields alone significantlyincreased the frequency of chromatid-type but not chromosome-typeaberrations in all cell lines examined, consistent with the finding thatTTFields cause DNA damage. These findings are depicted in FIG. 5D, whichshows that TTFields exposure for 48 h resulted in the induction ofchromatid-type aberrations in the panel of NSCLC lines; and in FIG. 5 E,which shows the frequency of chromosome-type aberrations after a 48 hTTFields exposure in combination with radiation. Note that in FIGS.5A-5E, the error bars represent the S.E.M. of three separate experimentsand asterisks represent a significant difference (P<0.05) betweenindicated conditions. It appears that the combined effect of TTFieldsplus IR increased both chromatid-type and chromosome-type aberrations,albeit not at a higher frequency than that of each agent alone.

TTFields Sensitize NSCLC Cells to IR: After observing the reduction inBRCA1 expression, a reduction in DNA DSB repair capacity, and increasedchromatid damage with TTFields exposure, the radioresponse of the panelof NSCLC cell lines was determined via clonogenic cell survival afterthe cells received either 2 or 4 Gy IR followed by TTFields treatmentfor 24, 48, and 72 h. The results of the inventors' evaluation ofradiosensitization effect of TTFields in combination with IR areprovided in Table 2 and FIG. 6. For this experiment, TTFields treatmentwas applied alone or immediately following treatment with 2 or 4 Gy of137Cs γ-rays. Survival was then assessed in all cell lines following 24,48, or 72 h of TTFields induction. Radiosensitization of TTFields wasevaluated by the Highest Single Agent approach for combinations of 2Gy+TTFields and 4 Gy+TTFields. All of the cell lines displayed anenhanced sensitivity to IR, although, consistent with earlier results,the degree of sensitization varied between cell lines. Note that in FIG.6, the error bars represent the S.E.M. of three separate experiments andasterisks represent values where survival was significantly decreased(P<0.05).

The inventors considered the combined effect of TTFields and IR to besynergistic if the combination index (CI) was >1 and the P-value was<0.05 for a given time point post IR and a given cell line (N=3) (seeMaterials and Methods). Based on these criteria, the combined effect of4 Gy IR and TTFields on cell death was found to be synergistic in allthe cell lines tested, whereas the combined effect of 2 Gy IR andTTFields on cell death was found to be synergistic in the H157, H4006,A549, and H1650 cell lines.

TABLE 2 Time CI CI Cell point (TTFields + P- (TTFields + P- line (h) 2Gy) value 4 Gy) value H157 24 1.48 <0.0001 2.23 0.001 H157 48 2.08<0.0001 1.14 0.003 H157 72 1.15 0.048 1.18 0.01 H4006 24 0.88 0.14 1.880.001 H4006 48 1.01 <0.0001 1.74 0.003 H4006 72 0.58 0.004 1.01 0.05A549 24 1.88 <0.0001 1.5 0.12 A549 48 1.14 0.001 2.36 <0.0001 A549 720.86 0.14 1.99 0.0007 H1650 24 1.48 0.17 1.19 0.64 H1650 48 1.21 <0.00010.9 0.21 H1650 72 1.35 <0.0001 1.47 0.03 H1299 24 0.91 0.68 3.32 <0.0001H1299 48 0.79 0.09 3.97 <0.0001 H1299 72 0.94 0.04 2.42 0.0003

TTFields Synergistically Enhanced the Cytotoxicity of the PARP InhibitorOlaparib: Experiments similar to those discussed above in connectionwith Table 2 and FIG. 6 were also conducted, but with exposure to IRreplaced with exposure to three different concentrations (either 10, 20,and 40 μM; or 12.5, 25, and 50 μM, depending on the cell line) of thePARP inhibitor Olaparib. More specifically, Olaparib at each of thedifferent concentrations was added to cells and the cells were thenexposed to TTFields for 30 or 60 h (or not exposed to TTFields for thecontrol) and immediately plated for survival. Baseline growth curves forexposing the various NSCLC cell lines to different concentrations ofOlaparib without TTFields are depicted in FIG. 7. The combination effectwas evaluated using the Highest Single Agent approach for variouscombinations and are listed in Table 3. Clonogenic assays are depictedin FIG. 8 for the different combinations of drug concentration andTTFields exposure time in Table 3.

The inventors considered the combined effect of TTFields and Olaparib tobe synergistic if the combination index (CI) was >1 and the P-value was<0.05 for a given time point and a given cell line. Based on thesecriteria and the data summarized in Table 3 below, the combined effectof TTFields and all tested concentrations of Olaparib on cell death wasfound to be synergistic in the H1299, H157, A549, and H4006 cell lines.

TABLE 3 Time TTFields + TTFields + TTFields + Cell point Olap 10 μM Olap20 μM Olap 40 μM line (h) CI P-value CI P-value CI P-value H1299 30 1.120.016 1.42 0.012 1.9 0.019 H1299 60 1.4 0.024 1.5 0.019 3.7 0.010 H15730 1.3 0.018 1.44 0.022 1.78 0.016 H157 60 1.63 0.022 1.43 0.023 1.680.020 Time TTFields + TTFields + TTFields + Cell point Olap 12.5 μM Olap25 μM Olap 50 μM line (h) CI P-value CI P-value CI P-value A549 30 1.120.031 1.46 0.021 1.32 0.015 A549 60 1.39 0.013 1.716 0.013 1.53 0.008H4006 30 1.17 0.03 1.17 0.02 1.32 0.02 H4006 60 1.13 0.02 1.34 0.02 1.960.01

The Triple Combination of TTFields, IR, and the PARP Inhibitor OlaparibSynergistically Enhances Cell Killing: Experiments similar to thosediscussed above in connection with Table 2 and FIG. 6 were alsoconducted for H1299 and H157 cell lines, both with and without the PARPinhibitor Olaparib. More specifically, Olaparib (at a concentration of20 μM) was added to cells, the cells were immediately exposed toTTFields for 24, 28, or 72 h, removed, irradiated (at different doses ofradiation), and immediately plated for survival.

The combination effect was evaluated using the Highest Single Agentapproach and are listed in Table 4 and depicted in FIGS. 9A and 9B,which summarize the clonogenic assays for the different combinations ofRT dosage and TTFields exposure time in the table, both with and withoutthe Olaparib.

The inventors considered the combined effect of TTFields and Olaparib tobe synergistic if the combination index (CI) was >1 and the P-value was<0.05 for a given time point and a given cell line. Based on thesecriteria and the data summarized in Table 4 below, the combined effectof TTFields, IR, and Olaparib on cell death was found to be synergisticin the H1299 and H157 cell lines.

TABLE 4 TTFields + TTFields + TTFields + TTFields + TTFields + Olap 1Gy2Gy Olap + 1Gy Olap + 2Gy Time CI P-value CI P-value CI P-value CIP-value CI P-value H1299 24 h 1.03 0.019 0.99 0.024 1.15 0.021 1.640.016 1.96 0.011 48 h 1.02 0.015 1.00 0.03 1.30 0.020 1.70 0.014 2.200.011 72 h 1.06 0.022 1.03 0.03 1.98 0.029 1.89 0.015 3.08 0.011 H157 24h 0.97 0.022 1.14 0.023 1.45 0.020 2.25 0.019 2.36 0.016 48 h 1.02 0.0140.90 0.015 1.55 0.015 2.12 0.015 2.07 0.008 72 h 1.49 0.016 0.92 0.0191.74 0.016 1.66 0.015 2.57 0.010

Discussion

The inventors confirmed that TTFields have antiproliferative effects,induce cell death, and alter the distribution of cells through the cellcycle, resulting in an enrichment of G2/M populations and the generationof a sub-G1 population indicative of apoptotic cells.

Earlier, Gera et al. showed that TTFields' sensitivity is dependent onp53 status in colon cancer cells; however, cell proliferation andsurvival results from the inventors' study and studies by others suggestthat TTFields' effects are independent of p53 status (Table 1 and FIG.1). Because the presence of a sub-G1 population and the increase in G2/Mcells are likely not sufficient to account for the differences insurvival observed when TTFields were applied across the NSCLC cellpanel, the inventors postulated that there are other novel mechanism(s)by which TTFields lead to cell killing. The inventors divided the NSCLCcell lines into two categories, that is, more responsive cell lines(H157 and H4006) and less responsive (A549, H1299, and H1650) celllines, and conducted gene expression analysis to understand the basisfor the differential response of NSCLC cell lines to TTFields.

The molecular basis of the differential responses to TTFields wasdemonstrated by supervised clustering analysis that clearly segregatedthe cell lines into a more responsive cluster and a less responsivecluster. To further elucidate the differences, the inventors comparedsignaling pathways involved in the genes that responded to TTFields ineach of the two cell line groups. The majority of the pathways werecommon in more responsive (15 out of 19 associated pathways) and lessresponsive cell lines (15 out of 27 associated pathways), which arerelated to cell cycle and DNA-damage response pathways. While thesepathways have been reported in previous studies, downregulation of theBRCA1 signaling pathway with TTFields exposure is a novel finding. Thefact that BRCA1 pathway downregulation is more pronounced in the moreresponsive cell lines than in the less responsive cell lines, evident bythe negative z-scores (FIG. 3A), suggests an inverted correlationbetween BRCA1 pathway activity and the sensitivity of cellular responseto TTFields.

BRCA1 together with BRCA2 have an important role in maintainingreplication fidelity through the repair of DSB damage by mediatinghomologous recombination and through non-homologous end joining during Sand G2 phases of cell cycle. DSBs can occur during IR exposure or asby-products of DNA replication. BRCA1 mutant mice exhibit chromosometranslocation and chromatid aberrations, and BRCA2 mutant miceaccumulate chromatid breaks and aberrant chromatid exchanges. BRCA1defects have been previously identified in multiple cancers includingbreast and pancreas. Defects in the BRCA genes predispose cells totherapeutics targeting single-strand break (SSB) repair pathways, suchas PARP inhibitors, resulting in what has been coined ‘syntheticlethality.’

On the basis of the inventors' findings, the inventors propose thatTTFields exposure may induce a conditional vulnerability, that is, theyinduce BRCAness because of the downregulation of the BRCA1 pathwaygenes. If this proposal is accurate, then TTFields could be applied incombination with PARP inhibitors without the potential for developingtherapy-resistant recurrent tumors as is common with molecularlytargeted therapies. This is supported by the inventors' results showingthe gradual accumulation of γ-H2AX foci following TTFields applicationover time and slowed DNA repair kinetics following IR exposure (γ-H2AXfoci and colocalized γ-H2AX and 53BP1 foci (FIGS. 5A-C). Indeed, themore responsive cell lines had, on average, more residual DNA repairfoci at 24 and 48 h post-IR than the less responsive cell lines, and Kimet al. also showed accumulation of γ-H2AX upon TTFields treatment. 53BP1localizes to DNA DSBs, which are physically distinct from DNAreplication stress, whereas γ-H2AX recruits MRE11, KU70, KU80 and RAD51to stalled replication forks at early time points. While not being boundby this theory, the inventors believe that TTFields not only slow downDNA damage repair kinetics but also induce replication stress based uponthe significant differences seen in colocalized γ-H2AX/53BP1 foci (FIGS.5A and 5C) and γ-H2AX foci alone (FIGS. 5B and C). Furthermore, theincreased frequency of chromatid-type aberrations (FIGS. 5D and 5E) isconsistent with ongoing replicative stress induced by TTFields because adefective response to replication stress leads to an accumulation ofchromatid-type aberrations. Hence, the inventors postulate that TTFieldsinduce replication stress and the reduction of BRCA1 pathway proteinsleads to an increased frequency of chromatid-type aberrations. Thenotion that TTFields induces DNA replication stress can explain both theincrease in DNA damage foci and the elevated frequency of chromatid-typeaberrations. Ongoing studies by the inventors' group are seeking tobetter understand the molecular underpinning of this induced replicationstress.

The reduced DNA DSB repair capacity seen in all cell lines when TTFieldswere applied post-IR is clearly linked to reduced cell survival (FIG. 6and Table 2). These data are consistent with findings reported by Kim etal. in which TTFields sensitized GBM cell lines to IR. In contrast tothe inventors' methods, these authors applied TTFields prior toirradiation, whereas the inventors first irradiated the cells and thenimmediately applied TTFields assuming that the chromosomal damagegenerated by IR would enhance the disruption of mitosis caused byTTFields exposure. Interestingly, both prior and post-TTFields treatmentsensitize cells to IR, which could have an impact on treatmentsequencing.

Turning next to the data discussed above in connection with FIGS. 7-9and Tables 3 and 4, it appears that TTFields downregulate the FANC/BRCApathways which are crucial for homologous recombination repair and whichresults in DNA replication fork stalling. PARP-1 is then hyperactivatedto reactivate stalled replication forks. PARP inhibitors block thisreplication fork re-activation. Furthermore, by downregulating BRCA2 viaTTFields and inhibition of PARP-1 activity, stalled replication fork DNAis no longer protected from Mre-11 degradation. By causing replicationstress and the downregulation of key genes associated with DNA repairand DNA replication fork protection or restart, TTFields generate asynergistic vulnerability to agents like PARP inhibitors.

The inhibition of DNA replication fork growth is supported by the datadepicted in FIG. 10, which shows that TTFields inhibit the increase inlength of newly replicated DNA fibers. More specifically, this datashows that there is no increase in newly replicated DNA when cells areexposed to TTFields for up to 60 h as measured by the DNA fiber assayusing halogenated nucleotides analogues in both H157 and H1299 cells.

Further support appears in FIG. 11, which shows that TTFields causereplication fork stress which results in R loop formation. Morespecifically, R-loops formed by TTFields exposure were quantified bydot-blot using a DNA-RNA hybrid specific S9.6 antibody in H157 and H1299cells. R loops, which are DNA:RNA hybrids are markers of DNA replicationfork stress and collapse that end in mitotic catastrophe. And FIG. 11shows that exposure of H157 and H1299 to TTFields increases R-loopformation.

In conclusion, TTFields induce a global antiproliferative and cytotoxiceffect on dividing cell populations; however, the relative sensitivityof cells to TTFields varies. These antitumor properties are due tomultiple mechanisms, likely acting in concert, that would suggestTTFields should be utilized as an adjuvant modality with RT and/or PARPinhibitors. Indeed, the inventors' data suggest by gaining additionalinsight into understanding the underlying molecular mechanisms governingTTFields' antitumor effects optimizing combinatorial strategies ofTTFields, IR and/or PARP inhibitors in preclinical models isappropriate. At this junction PARP inhibitors are particularlyattractive, based on the data set forth herein. Lastly, whether specificmolecular signatures as reported in this study will predict whichpatients will respond better to TTFields is worth exploring clinicallywhere possible.

Materials and Methods

Cell Culture: Human NSCLC cell lines (H157, H4006, A549, H1299 andH1650) were purchased from American Tissue Culture Collection. All thesecell lines were grown in RPMI medium supplemented with 10% (v/v) fetalbovine serum (Atlanta Biologicals, Flowery Branch, Ga., USA) andpenicillin/streptavidin (final concentration 50 μg/ml; Sigma-Aldrich,St. Louis, Mo., U29SA). All cells were grown at 37° C. in a humidifiedincubator constantly supplied with 5% CO₂.

Tumor Treating Fields: The inventors used the inovitro system (NovoCureLtd, Haifa, Israel) to generate TTFields that use two pairs ofelectrodes printed perpendicularly on the outer walls of a Petri dishcomposed of high dielectric constant ceramic (lead magnesiumniobate-lead titanite (PMN-PT)). The transducer arrays were connected toa sinusoidal waveform generator that generate low-intensity electricfields at the desired frequencies in the medium as summarized in Table 1above. The orientation of the TTFields was switched 90° every 1 s, thuscovering the majority of the orientation axis of cell divisions, aspreviously described by Kirson et al. Plate temperature was maintainedat 37° C. by placing the plates in a refrigerated incubator where thetemperature was maintained at 19° C. to dissipate the heat generated bythe inovitro system. The temperature was measured by 2 thermistors(Omega Engineering, Stamford, Conn., USA) attached to the ceramic walls.All cell suspensions were grown on a cover slip inside the inovitro dish(NovoCure Ltd) and treated with TTFields for the times indicated in thefigure legends.

Cell Growth Assay: Human NSCLC (H157, H4006, A549, H1299 and H1650) celllines were treated with different frequencies of TTFields indicated for24, 48 and 72 h, and cell growth was counted using a Beckman coultercounter (Beckman Coulter Inc, Indianapolis, Ind., USA) in triplicatesfor each sample. Growth curve graphs were drawn using the average cellnumber counted at each time point and the given TTFields frequency usingGraphPad Prism V.6 (GraphPad Software Inc, La Jolla, Calif., USA).

Cell Cycle Analysis: Cells at specific times and treatments wereharvested and fixed in 75% ice-cold ethanol at −20° C. for 24 h. Fixedcells were washed with PBS and incubated in 500 μl of PI stainingsolution, that is, PBS containing 1 mg/ml RNAse A (Sigma-Aldrich), 0.05%triton X-100 and 30 μg/ml of PI (Sigma-Aldrich) for 30 min at 37° C. Thecell cycle distribution was determined using a FACSCalibur system (BDBiosciences, San Jose, Calif., USA). More than 10,000 cells per samplewere counted and the results were analyzed using FlowJo software v8.7.1(Tree Star Inc, Ashland, Oreg., USA).

Labeling and Hybridization of RNA for Gene Expression Analysis: IlluminaWhole Genome HumanWG6 v4 Expression BeadChips (Illumina Inc, San Diego,Calif., USA) were used. Each RNA sample (0.5 μg) was amplified using theIllumina TotalPrep RNA amplification kit with biotin UTP (Enzo LifeSciences, Inc., Farmingdale, N.Y., USA) labeling. T7 oligo(dT) primerswere used to generate single-stranded cDNA followed by a second-strandsynthesis to generate double-stranded cDNA, which is thencolumn-purified. In vitro transcription was done to synthesizebiotin-labeled cRNA using T7 RNA polymerase. The cRNA was thencolumn-purified and checked for size and yield using the Bio-RadExperion system (Bio-Rad Laboratories, Hercules, Calif., USA). cRNA (1.5μg) was then hybridized for each array using standard Illumina protocolswith streptavidin-Cy3 (Amersham Biosciences, Piscataway, N.J., USA)being used for detection. Slides were scanned on an Illumina Beadstation(Illumina Inc).

Data Processing and Significance Analysis of Differential GeneExpression: Summarized expression values for each probe set weregenerated using BeadStudio 3.1 (Illumina Inc). The data werebackground-subtracted and quantile-quantile-normalized across samplesusing the MBCB algorithm. Normalized gene expression values were used togenerate plots for comparisons. Analysis of differentially expressedgenes in treated cell lines was performed using SAM. FDR<0.05 wasconsidered to be statistically significant. Clustering analysis andheatmaps were generated using the Partek Genomic Suite software (PartekIncorporated, St. Louis, Mo., USA). Gene ontology and pathway analysiswas performed using IPA (QIAGEN, Redwood City, Calif., USA).

Immunoblotting: Laemmli sample buffer (4×; Bio-Rad Laboratories) wasadded to 30 μs of each protein sample and the mixtures were boiled at95° C. for 10 min. Protein mixtures were then loaded on 10% SDS-PAGE gelfollowed by transfer to PVDF membrane for 1 h at 90 V at 4° C. Themembrane was blocked with 5% fat-free milk in PBST for 1 h at roomtemperature and probed with anti β-actin (1:5000; Cell Signaling,Danvers, Mass., USA), anti-BRCA1 (1:1000), anti-FANCD2 (1:2000) andanti-FANCA (1:500; Novus Biologicals LLC, Littleton, Colo., USA) in PBSTcontaining 2% bovine serum albumin (Thermo Fisher Scientific Inc,Bridgewater, N.J., USA) overnight at 4° C. Membranes were washed withphosphate-buffered saline with 0.1% Tween-20 (PBST; 3×, 10 min, each)followed by incubation with secondary antibodies (1:5000) conjugatedwith horseradish peroxidase (GE Healthcare, Buckinghamshire, UK) for 1 hat room temperature. Membranes were developed using a chemiluminescencedetection kit (Thermo Scientific, Rockford, Ill., USA) on FluorChem Msystem (ProteinSimple, San Jose, Calif., USA). Quantification was doneusing the ImageJ software (NIH, Bethesda, Md., USA) and normalized usingthe corresponding actin density.

Immunofluorescence: Cells were seeded on glass coverslips and aftertreatment cells were washed and fixed with ice-cold methanol. Thesamples were blocked with 10% normal goat serum for 1 h and incubatedwith phospho-histone-γ-H2AX antibody (Ser139; Upstate Biotechnology,Temecula, Calif., USA) and p53-binding protein 1 (53BP1) antibody (CellSignaling). Samples were washed three times for 5 min in PBS, and thenincubated with Alexa Fluor 488-conjugated antirabbit antibody and AlexaFluor 555-conjugated anti-mouse antibody (Invitrogen, Carlsbad, Calif.,USA) for 1 h. Nuclei were counterstained with DAPI contained inVecatshield mounting medium (Vector Laboratories Inc, Burlingame,Calif., USA). The stained cells were then analyzed under a fluorescencemicroscope (Axio Imager M2, Carl Zeiss, Thornwood, N.Y., USA) with a ×63objective (oil immersion, aperture 1.3) with five slices of z-stacks of0.2 μM thickness each. Quantitative image analysis of 40 nuclei fromeach experiment was performed using Cell module in Imaris softwareversion 8.0 (Bitplane, Concord, Mass., USA).

Cytogenetic Analyses: Preparation of metaphase chromosome spreads andcytogenetic analysis were performed. Briefly, cultured cells weretreated with 1 μM colcemid solution (Thermo Scientific) for 3-4 h at 37°C., trypsinized, incubated for 30 min in a hypotonic solution of 75 mMKCl solution and subsequently fixed with 3:1 methanol to acetic acid.Samples were then dropped on to glass slides and stained with either 5%Giemsa (Sigma-Aldrich) or prolong antifade gold reagent with DAPI (LifeTechnologies, Carlsbad, Calif., USA) for scoring. The presence ofchromosome-type aberrations (deletions, dicentric chromosomes and rings)and chromatid-type (gaps, breaks, deletions, and radial chromosomearrangements) was detected under a microscope (Axio Imager M2, CarlZeiss) and ˜30 metaphase cells per treatment group were scored andaverages displayed as the frequency of aberrations per cell.

Radiation Exposure and Clonogenic Cell Survival: To study the effect ofradiation sensitivity on NSCLC cells, exponentially growing cells weretreated with IR using a Mark II Cs irradiator (J L Shepherd andAssociates) at a dose rate of 3.47 Gy/min, followed by immediateapplication of TTFields for 24, 48 and 72 h. Cells were then re-seededinto 60 mm dishes and incubated for up to 2 weeks. Colonies containing50 or more cells were considered viable. The data are presented as themean±S.E.M. of three independent experiments. The radiosensitizationeffect of TTFields was evaluated according to The Highest Single Agentapproach by calculating the CI as given below.

CI=SF _(IR) ×SF _(TTFields))/SF _(IR+TTFields)

Where SF=Survivalfraction

The combination effect was considered enhanced/synergistic when CI>1,additive when CI=1. Statistical significance for a positive effect wasdetermined by the P-value of a two-way ANOVA multiple comparisonstatistical test comparing the combination (TTFields plus IR) to thesingle agent showing the greatest cell killing for a given dose and timeafter IR.

For the Triple Combination of TTFields, IR, and Olaparib, CI wascalculated using the following equations:

CI(TTFields+olap)=(SF _(Olap) ×SF _(TTFields))/SF _(Olap+TTFields)

CI(TTFields+1 Gy)=(SF _(1Gy) ×SF _(TTFields))/SF _(1Gy+TTFields)

CI(TTFields+2 Gy)=(SF _(2Gy) ×SF _(TTFields))/SF _(2Gy+TTFields)

CI(TTFields+olap+1 Gy)=(SF _(1Gy+Olap) ×SF _(TTFields))/SF_(Olap+TTFields+1Gy)

CI(TTFields+olap+2 Gy)=(SF _(2Gy+Olap) ×SF _(TTFields))/SF_(Olap+TTFields+2Gy)

Where SF=Survival fraction

CONCLUSION

The in vitro experiments described above demonstrate that applyingTTFields in combination with a PARP inhibitor provides a synergisticeffect against NSCLC cells; and that TTFields in combination with bothIR and a PARP inhibitor also provides a synergistic effect against NSCLCcells.

Note that although the examples discussed herein uses Olaparib incombination with TTFields, in alternative embodiments other PARPinhibitors (including but not limited to Rucaparib and Niraparib) may beused in place of Olaparib. Note also that while the experimental resultsdescribed herein were obtained in vitro, the inventors expect that theywill carry over to the in vivo context.

These results establish that cancer cells can be killed by delivering aPARP inhibitor to the cancer cells and applying an alternating electricfield with a frequency between 80 and 300 kHz to the cancer cells. Atleast a portion of the applying step is performed simultaneously with atleast a portion of the delivering step. Note that in the in vitrocontext, the delivery of the PARP inhibitor to the cancer cells occurscontinuously from a first time (t₁) when the PARP inhibitor isintroduced into the container that is holding the cancer cells untilsuch time (t₂) as the PARP inhibitor is removed or exhausted. As aresult, if TTFields are applied to the cancer cells between t₁ and t₂,the applying step will be simultaneous with at least a portion of thedelivering step. In the in vivo context, the delivery of the PARPinhibitor to the cancer cells occurs continuously from a first time (t₁)when the PARP inhibitor is circulating in the patient's body (e.g.,after administering it systemically) or introduced into the vicinity ofthe cancer cells until such time (t₂) as the PARP inhibitor iseliminated from the patient's body or exhausted. As a result, ifTTFields are applied to the cancer cells between t₁ and t₂, the applyingstep will be simultaneous with at least a portion of the deliveringstep.

Examples of PARP inhibitors that may be used for the methods describedherein include Olaparib, Rucaparib, and Niraparib. The optimal frequencyand field strength will depend on the particular type of cancer cellbeing treated. For certain cancers, the optimum frequency will bebetween 100 and 200 kHz and the field strength will be at least 1 V/cm.In some embodiments, a deviation from the optimum frequencies may bemade. For example, when the optimum frequency for treatment is 100 kHz,frequencies down to 80 kHz may still be effective. Or when the optimumfrequency for treatment is 200 kHz, frequencies up to 300 kHz may stillbe effective.

In those embodiments that include radiation treatment, the radiationtreatment may be performed before the applying step has begun, after theapplying step has ended, or while the applying step is ongoing.

While the present invention has been disclosed with reference to certainembodiments, numerous modifications, alterations, and changes to thedescribed embodiments are possible without departing from the sphere andscope of the present invention, as defined in the appended claims.Accordingly, it is intended that the present invention not be limited tothe described embodiments, but that it has the full scope defined by thelanguage of the following claims, and equivalents thereof.

1.-19. (canceled)
 20. A method of killing cancer cells selected from thegroup consisting of breast cancer cells, pancreatic cancer cells,glioblastoma cells, and ovarian cancer cells, comprising: delivering aPARP inhibitor to the cancer cells; and applying an alternating electricfield to the cancer cells, the alternating electric field having afrequency between 80 and 300 kHz.
 21. The method of claim 20, whereinthe cancer cells are breast cancer cells.
 22. The method of claim 20,wherein the PARP inhibitor comprises olaparib.
 23. The method of claim20, wherein at least a portion of the applying step is performedsimultaneously with at least a portion of the delivering step.
 24. Themethod of claim 20, wherein the applying step has a duration of at least72 hours.
 25. The method of claim 20, wherein the frequency of thealternating electric field is between 100 and 200 kHz.
 26. The method ofclaim 20, wherein the alternating electric field has a field strength ofat least 1 V/cm in at least some of the cancer cells.
 27. The method ofclaim 26, wherein the applying step has a duration of at least 72 hoursand wherein the frequency of the alternating electric field is between100 and 200 kHz.
 28. The method of claim 20, wherein the concentrationof the PARP inhibitor is from about 10 to about 50 μM.
 29. The method ofclaim 28, wherein the PARP inhibitor is olaparib.
 30. A method ofkilling cancer cells selected from the group consisting of breast cancercells, pancreatic cancer cells, glioblastoma cells, and ovarian cancercells, comprising: delivering a PARP inhibitor to the cancer cells;applying an alternating electric field to the cancer cells, thealternating electric field having a frequency between 80 and 300 kHz;and treating the cancer cells with a radiation therapy.
 31. The methodof claim 30, wherein the cancer cells are breast cancer cells.
 32. Themethod of claim 30, wherein the PARP inhibitor comprises olaparib. 33.The method of claim 30, further comprising repeating the delivering,applying, and treating steps at least five times.
 34. The method ofclaim 33, wherein each repetition of the treating step comprisesdelivering at least 2 Gy of radiation to a target area.
 35. The methodof claim 33, wherein each repetition of the treating step comprisesdelivering at least 4 Gy of radiation to a target area.
 36. The methodof claim 33, wherein the frequency of the alternating electric field isbetween 100 and 200 kHz.
 37. The method of claim 33, wherein thetreating step is performed immediately after the applying step in eachrepetition.
 38. The method of claim 33, wherein the applying step isperformed immediately after the treating step in each repetition. 39.The method of claim 30, wherein the concentration of the PARP inhibitoris from about 10 to about 50 μM.
 40. The method of claim 39, wherein thePARP inhibitor is olaparib.
 41. A method of killing cancer cells havingdownregulated BRCA1 pathway signaling compared to normal cells, themethod comprising: delivering a PARP inhibitor to the cancer cells; andapplying an alternating electric field to the cancer cells, thealternating electric field having a frequency between 80 and 300 kHz.